Identification of substances that inhibit NEMO oligomerization
a technology of nemo and oligomerization, which is applied in the field of identification of substances that inhibit nemo oligomerization, can solve the problems of severe defects in nf-b activation, cell lacking nemo is unable to assemble the ikk complex, etc., and achieves the effects of inhibiting ikk complex formation or activation, inhibiting ikk complex formation, and inhibiting nemo oligomerization
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[0051]In the following experiments, we reevaluated the molecular mass and the activity of the IKK complex in extracts from resting or Tax-activated cells by glycerol gradient ultracentrifugation since this method alleviates the problems encountered with NEMO in gel filtration. We also monitored a stimulus-dependent oligomerization of NEMO in living cells through FRET experiments.
[0052]Methods
[0053]Cell Culture and Transient Transfections
[0054]Rat-1, M319-5b (see supplementary data), 5R fibroblast and wild type, Ikkα− / −Ikkβ− / − and Nemo− / − MEF cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serumn. For 5R cells, 20% 5R conditionned medium was added to fresh culture medium. Transfections using Fugene 6 (Roche) were performed according to the manufacturer protocol using, per well of a 6 well-plate, 2 μg of pECFP-Nemo or pEYFP-Nemo plasmid (single transfection) or 1 μg each (double transfection). For plasmid obtaining, see supplementary data. In ...
example 2
[0078]Plasmids
[0079]The murine Nemo gene was amplified using plasmid HA-Nemo (Agou, F., et al (2002) J Biol Chem, 277, 17464-17475) as template (EcoR1 and BamH1 site extended primer sequences on request). The PCR product, pECFP-C1 and pEYFP-C1 plasmids (BD Biosciences) were digested by BamH1 and EcoR1 and the digested PCR product was ligated to the digested plasmids giving rise to pECFP-Nemo and pEYFP-Nemo vectors. The cfp / yfp-Nemo regions of the plasmids were sequenced to control gene integrity.
[0080]Characterization of the M319-5b Cells
[0081]M319-5 cells were derived from rat fibroblast transformed by a mutant Tax protein competent to activate NF-κB. As M319-5 cells tend to loose Tax expression after extended passages in the presence of G418, stable transfection of M319-5 cells with Igκ2bsrH have been performed to confer them a NF-κB dependent blasticidin S resistance (Yamaoka, S.,et al (1998) Cell, 93, 1231-1240). The resistant M319-5 cells (M319-5b) express the Tax protein (FIG....
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